1. Field of the Invention
The present invention relates to a method for preparation of nephritogenoside, a substance having an ability to induce glomerulonephritis in animals. More specifically, the invention relates to a method for effectively and readily preparing nephritogenoside from some organs of various animals including human being and especially from human placenta at low cost.
2. Description of the Prior Art
Nephritogenoside is a glycopeptide having a structural formula shown in the attached FIG. 1. The glycopeptide has been isolated from rat kidney by the present applicant while he made investigations to purify an antigen of Masugi's glomerulonephritis. The glycopeptide has a specific ability that a single injection of the substance to a homologous or heterologous animal can induce glomerulonephritis in the animal without the aid of its immunological mechanism, and the induced glomerulonephritis will lead to contracted kidney without fail. This is the same progress as can be observed in human chronic glomerulonephritis. In addition, the substance can be extracted from tissues of normal animals (including healthy human being) and has a specificity that it induces glumerulonephritis in homologous animals.
The glycopeptide is expected, therefore, to perform a very important part in studying possible causes and therapeutic treatments of human glomerulonephritis. It also arrests much attention of medical and pharmaceutical fields to its wide applications and potential development of a therapeutic agent for human glomerulonephritis which may be obtained by making some modifications in the structure of nephritogenoside.
The previous investigations made by the applicant revealed that nephritogenoside exists not only in rats but in other animals such as human being, dogs, rabbits, cattles and pigs and that it is widely distributed throughout the body including lung, heart, aorta and placenta rather than only in kidney. This fact can be explained by its presence in basement membrane of microvessels which exist in those organs in a large quantity.
A previous method for preparation of nephritogenoside is well known to those skilled in the art.
According to that method, which was disclosed in U.S. Pat. No. 4,382,887 by the present applicant, nephritogenoside can be prepared by using kidney excised from rat.
More specifically, renal cortex is first excised from rat kidney to collect glomeruli and thus collected glomeruli are sonicated to collect glomerular basement membrane (GBM). The GBM is digested with trypsin for a short time (3 hours) and centrifuged to take the supernatant liquid, which is then dialyzed and lyophilized. Thus obtained powder is further digested with different proteinases (for example, trypsin, collagenase, and pronase) in succession to a possible minimum size and ultracentrifuged to take the supernatant liquid, which is then dialyzed and lyophilized. Next, the substance obtained is subjected to zone electrophoresis to collect only the first half of the glycopeptide peak exhibiting anthrone reaction. The collected substance is treated with trichloroacetic acid to take the supernatant liquid, which is dialyzed and separated through SEPHADEX G 200 or BIOGEL P 300 column chromatography. Of the two sugar peaks obtained, void volume fractions are collected for dialysis, and components which remain in the dialysis membrane are desalted and condensed to be subjected to DEAE cellulose column chromatography for purification.
Alternatively, a chemical synthesis method has been developed (Japanese Patent Publications No. 11598/84 and No. 26398/85) to prepare the same substance. According to it, glucose is bonded in sequence to produce the sugar moiety of the above glycopeptide and then the peptide moiety is bonded to the sugar moiety through N-glycoside bonding.
The first previous method in which rat kidney is used as the starting material adopts very complicated steps for preparation, and yields only 5 to 6 mg of nephritogenoside from kidneys of 1,200 rats. This low yield and resultant economic problems were one of the subjects the applicant must continue to study.
Also the second previous method to prepare the glycopeptide through chemical synthesis has a complicated step to bond glucose which comprises the sugar moiety. In addition, on forming N-glycoside bonding between the sugar chain moiety and peptide chain moiety, .beta.-bonds besides desired .alpha.-bonds are formed. When removing all products having the .beta.-bonds, some .alpha.-bond losses cannot be avoided, resulting in a low yield.